WO2022226079A1 - Anticorps neutralisants contre le sars-cov-2 - Google Patents

Anticorps neutralisants contre le sars-cov-2 Download PDF

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Publication number
WO2022226079A1
WO2022226079A1 PCT/US2022/025560 US2022025560W WO2022226079A1 WO 2022226079 A1 WO2022226079 A1 WO 2022226079A1 US 2022025560 W US2022025560 W US 2022025560W WO 2022226079 A1 WO2022226079 A1 WO 2022226079A1
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seq
antigen
antibody
binding fragment
amino acid
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PCT/US2022/025560
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English (en)
Inventor
Syamal Raychaudhuri
Gregory C. Ireton
Lihong Li
Sriram JAKKARAJU
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Inbios International, Inc.
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Publication of WO2022226079A1 publication Critical patent/WO2022226079A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the disclosure relates to antibodies directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as related methods of use.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Coronavirus disease 2019 is the infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 is a highly infectious novel coronavirus that causes upper respiratory tract infections that can quickly develop into more serious conditions.
  • SARS-CoV-2 infection can prove fatal, although a majority of infected individuals, especially those in younger age groups, remain asymptomatic or display mild symptoms.
  • the disclosure relates to antibodies, and antigen-binding fragments thereof, that bind to a SARS-CoV-2 Spike protein, and uses thereof.
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) Spike protein, or a fragment thereof, comprising: a heavy chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively; and/or a light chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively; or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid modifications, e.g., substitutions, in each of said CDR sequences.
  • the variant comprises less than 8 amino acid modifications in all CDR sequences combined.
  • the variant comprises less than 7, less than 6, less than 5, less than
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) Spike protein, or a fragment thereof, comprising: a heavy chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 respectively; and/or a light chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 respectively; or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid modifications, e.g., substitutions, in each of said CDR sequences.
  • the variant comprises less than 8 amino acid modifications in all CDR sequences combined.
  • the variant comprises less than 7, less than 6, less than 5, less than 4,
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) Spike protein, or a fragment thereof, comprising: a heavy chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 respectively; and/or a light chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 respectively; or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, e.g., substitutions, wherein said variant comprises less than 8 amino acid modifications, e.g., substitutions, in each of said CDR sequences.
  • the variant comprises less than 8 amino acid modifications in all CDR sequences combined.
  • the variant comprises less than 7,
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) Spike protein, or a fragment thereof, comprising a heavy chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 respectively; and/or a light chain polypeptide, or fragment thereof, comprising three CDRs comprising the amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 respectively; or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid modifications, e.g., substitutions, in each of said CDR sequences.
  • the variant comprises less than 8 amino acid modifications in all CDR sequences combined.
  • the variant comprises less than 7, less than 6, less than 5, less than 4, less
  • the antibody, or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ IDNOs: 25, 27, 29 or 31, optionally comprising the three CDRs ofthe417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E heavy chain.
  • the antibody, or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 25, 27, 29 or 31.
  • the antibody, or fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 26, 28, 30 or 32, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E light chain.
  • the antibody, or fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 26, 28, 30 or 32.
  • the isolated antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:26.
  • the isolated antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 28.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28.
  • the isolated antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 29 and a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 30.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:29, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:30.
  • the isolated antibody, or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 32.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:32.
  • the antibody has an IgG format, e.g., an IgG1 or IgG2 format, e.g., an IgG2a or IgG2b format.
  • the antibody comprises an Fc domain, framework sequences, and/or other domains of a full antibody, e.g., an IgG formatted antibody.
  • the antibody, or antigen-binding fragment thereof is humanized.
  • the antibody, or antigen-binding fragment thereof is an IgG antibody, a single chain antibody, a single chain variable fragment (scFv), a univalent antibody lacking a hinge region, a VHH or single-domain antibody (sdAb), or a minibody.
  • the antibody, or antigen-binding fragment thereof is formatted as an IgG antibody.
  • the antibody, or antigen-binding fragment thereof comprises two heavy chain polypeptides, or fragments thereof, and two light chain polypeptides, or fragments thereof, disclosed herein.
  • the heavy chain polypeptides, or fragments thereof are covalently bonded to each other, and is some embodiments, the light chain polypeptides, or fragments thereof, are each covalently bonded to a heavy chain polypeptide or fragment thereof.
  • the antibody, or antigen-binding fragment thereof is a VHH or sdAb.
  • the antibody, or antigen-binding fragment thereof is a Fab or a Fab’ fragment.
  • the antibody, or antigen-binding fragment thereof binds to a Spike protein comprising SEQ ID NO: 43, or a fragment or variant thereof. In some embodiments of any of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure, the antibody, or antigen-binding fragment thereof, binds to a Spike protein comprising SEQ ID NO: 44, or a fragment or variant thereof. In some embodiments of any of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure, the antibody, or antigen- binding fragment thereof, binds to a Spike protein comprising SEQ ID NO: 45, or a fragment or variant thereof.
  • the variant comprises a Spike protein sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the Spike protein of SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45.
  • the isolated antibody, or an antigen-binding fragment thereof competes with any one of the antibodies of the disclosure for binding to a Spike protein or fragment thereof, optionally the Spike protein comprising SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45, or a fragment or variant thereof.
  • the isolated antibody, or an antigen-binding fragment thereof binds to the Spike protein with a Kd value of 50 mM or lower.
  • the isolated antibody, or an antigen-binding fragment thereof inhibits Spike protein interaction with a human ACE2 receptor.
  • the isolated antibody, or an antigen-binding fragment thereof binds an epitope in the receptor binding domain (RBD) of the Spike protein.
  • the epitope is linear or non-linear.
  • the epitope comprises or overlaps with the binding site of a human ACE2 receptor on the Spike protein.
  • the isolated antibody, or an antigen-binding fragment thereof neutralizes SARS-CoV-2.
  • the isolated antibody, or an antigen-binding fragment thereof inhibits viral and cell membrane fusion.
  • the isolated antibody, or an antigen-binding fragment thereof inhibits viral infectivity.
  • the disclosure provides methods for producing an isolated antibody, or antigen-binding fragment thereof, disclosed herein.
  • the isolated antibody, or an antigen-binding fragment thereof is produced in a mammalian cell or a plant.
  • the isolated antibody, or an antigen-binding fragment thereof is produced in a mammalian cell.
  • the disclosure provides an isolated polypeptide comprising a heavy chain, a heavy chain variable region, or a fragment thereof, disclosed herein; or an isolated polypeptide comprising a light chain, a light chain variable region, or a fragment thereof, disclosed herein.
  • the disclosure provides an isolated polynucleotide encoding any one of the isolated antibodies, or a heavy chain or light chain or antigen-binding fragment thereof, of the disclosure.
  • the disclosure provides an expression vector comprising any one of the isolated polynucleotides of the disclosure.
  • the disclosure provides an isolated host cell comprising any one of the expression vectors of the disclosure.
  • the disclosure provides a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier, and a therapeutically effective amount of any one of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure.
  • the disclosure provides a method for preventing, inhibiting, reducing, or treating a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, comprising administering to a subject an effective amount of any one of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure, optionally in a pharmaceutical composition.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides a method for preventing, inhibiting, reducing, or treating a disease or disorder caused by or associated with severe acute respiratory syndrome coronavirus 2 (S ARS-CoV -2), comprising administering to a subj ect a therapeutically effective amount of any one of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure, optionally in a pharmaceutical composition.
  • S ARS-CoV -2 severe acute respiratory syndrome coronavirus 2
  • the subject is human.
  • the administration of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition, of the disclosure generates an immune response against SARS-CoV-2.
  • the immune response comprises antibody-dependent cell cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP).
  • the method further comprises administering to the subject an anti-viral drug, a viral entry inhibitor, or a viral attachment inhibitor.
  • the method further comprises administering to the subject a booster dose comprising the isolated antibody, or antigen-binding fragment thereof, of the disclosure.
  • the pharmaceutical composition comprises two or more antibodies specific to SARS-CoV-2.
  • the pharmaceutical composition is administered prior to or after exposure to SARS-CoV-2.
  • the pharmaceutical composition is administered at a dose sufficient to neutralize SARS-CoV-2.
  • the disclosure provides a method of determining if a subject is infected with SARS-CoV-2, comprising: contacting a biological sample obtained from the subject with any one of the isolated antibodies, or antigen-binding fragments thereof, of the disclosure; determining an amount of the antibody, or antigen-binding fragment thereof, bound to the biological sample, thereby determining the presence or absence of SARS-CoV-2 in the biological sample, thus determining if the subject is infected with SARS-CoV-2.
  • FIGs. 1A and IB are schematic diagrams depicting the SARS-CoV-2 virus and neutralizing antibodies.
  • FIG. 1A depicts the SARS-CoV-2 virus surface covered by Spike protein (S) trimers and
  • FIG. IB depicts neutralizing antibodies that can bind and block the ACE2 receptor binding domain of the Spike protein.
  • FIG. 2 is a schematic drawing depicting an in vitro ELISA assay (Competition ELISA) to measure inhibition of S-ACE2 interaction by Abs using recombinant Spike proteins (S).
  • Step 1 depicts the addition of Ace2Fc protein in the presence or absence of Abs to wells coated with S.
  • Step 2 depicts the detection of Ace2Fc interaction with S by an anti-Fc antibody conjugated to horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • Step 3 depicts G color intensity measured at OD450 nm as an indicator of Ace2-S binding levels in the well.
  • FIGs. 3A-3D are graphs depicting the ability of Abs to inhibit binding of S with ACE2Fc by Competition ELISA.
  • OD450 nm measures the binding of 100 ng/mL ACE2Fc to S coated on the wells in the presence of the indicated final concentrations of various Abs on the X axis.
  • FIG. 3 A depicts competition binding in the presence of IgG1 Abs with the lowest line being 417.66D
  • FIG. 3B depicts competition binding in the presence of IgG2a Abs with the lowest line being 417.19C and the second lowest line being 417.53C;
  • FIG. 4D depicts binding of ACE2Fc to S in the absence of Abs.
  • FIG. 4 is a graph depicting percent inhibition (Y axis) by seven Abs that inhibited S- ACE2Fc binding by competition ELISA as a function of Ab concentration (X axis).
  • FIGs. 5A and 5B are a pair of graphs depicting the performance of Abs in neutralizing SARS-CoV-2 pseudo virus infection of 293T cells expressing ACE2 on the surface in a Pseudovirus Neutralization assay.
  • BREP-013 and JC 2007 are positive and negative control antibodies, respectively.
  • FIG. 5A depicts % neutralization at l ⁇ g/mL concentrations, wherein 7 Abs displayed > 50% neutralization indicating strong inhibition of viral entry into the cells.
  • FIG. 5B depicts the titration of the seven neutralizing Abs plotted for inhibition curves to estimate IC50 concentrations.
  • FIG. 6 provide a set of graphs showing the measurement of the ability of Abs to inhibit binding of S protein with ACE2Fc by Competition ELISA.
  • OD450 nm measures the binding of 100 ng/mL ACE2Fc to S protein coated on the wells in the presence of the indicated final concentrations of Abs.
  • Competition biding in the presence of wild type Spike protein (Left); Delta variant Spike protein (Middle) and Omicron variant Spike protein (Right) are depicted.
  • FIG. 7 is a graph showing percent inhibition of wild type, Delta and Omicron S- ACE2Fc binding by competition ELISA as a function of 417.19C concentration.
  • antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have sub-classes as well, such as IgG1, IgG2, IgG3, IgG4.
  • the antibodies that neutralize infection by SCoV2 can belong to various kinds of antibody isotypes.
  • the neutralizing antibodies can be IgG1, IgG2 (e.g., IgG2a and IgG2B), IgG3 and/or IgG4 isotype antibodies.
  • the antibodies, and antigen-binding fragments disclosed herein may be IgG, IgM, IgA, IgE or IgD, or any of IgG1, IgG2 (e.g., IgG2a and IgG2B), IgG3 and/or IgG4 isotype antibodies.
  • the light chain may be a Kappa chain or a Lambda chain.
  • the antibodies, and antigen-binding fragments disclosed herein may be Kappa or Lambda light chain antibodies.
  • antigen binding site refers to the part of the immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light (“L”) chains.
  • V N-terminal variable
  • H heavy
  • L light
  • FR framework regions
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three- dimensional space to form an antigen binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity- determining regions," or "CDRs.”
  • CDRs complementarity- determining regions
  • antibody may refer to immuno-globulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Ig immunoglobulin
  • antibodies specifically binds or “immunoreacts with” it is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not significantly react with other, unrelated polypeptides.
  • Antibodies may include, but are not limited to, polyclonal, monoclonal, and chimeric antibodies.
  • epitopic determinants may include any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor.
  • Epitopic determinants may consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide.
  • immunological binding and “immunological binding properties” may refer to the non-covalent interactions which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. Certain methods entail measuring the rates of antigen-binding site/ antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • K on the "on rate constant”
  • Koff the "off rate constant”
  • K on the concentrations and the actual rates of association and dissociation.
  • the ratio of Koff / Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant K d .
  • K d is calculated using biolayer interferometry (BLI) measurements. BLI detects the surface changes on biosensor tips induced by protein-protein association and dissociation by using analyzing the interference pattern of white light reflected from the surface.
  • K d is calculated using surface plasmon resonance (SPR), which detects the reflectivity changed induced by molecular adsorption, such as polymers, DNA or proteins, etc. by changes in reflection angles.
  • SPR surface plasmon resonance
  • a SARS-CoV-2 protein or a derivative, fragment, analog, homolog or ortholog thereof may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • Human angiotensin-converting enzyme 2 (ACE2) is the established functional receptor by which SARS-CoV-2 enters host cells.
  • the corresponding viral protein is the trimeric Spike protein (S) that is expressed on the surface of SARS-CoV-2.
  • S is a protein expressed by all known coronaviruses with varying degrees of sequence conservation. S is the chief architect of membrane fusion and host cell entry, by means of its affinity for ACE2 and subsequent processes that enable infection.
  • SI contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of ACE2.
  • RBD receptor binding domain
  • PD peptidase domain
  • the interaction of virus surface S protein with the ACE2 receptor on the host mucosal cell surface facilitates entry and infection of cells by SARS-CoV-2.
  • a neutralizing antibody specific to S protein may be able to inhibit this interaction significantly.
  • the SCov-2 Spike protein has been mutating and evolving continuously, it is desirable to generate Abs that can bind and inhibit S proteins that have been characterized in variants of concern.
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to the Spike protein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant.
  • the variant has high transmissibility and virulence.
  • the variant is the SARS-CoV-2 variant as defined by the Centers for Disease Control and Prevention (see https://www.cdc.gov/coronavirus/2019-ncov/variants/about-variants.html).
  • the variant is the SARS-CoV-2 Delta strain (e.g., B.1.617.2) or a subvariant thereof.
  • the variant is the SARS-CoV-2 Omicron strain (B.1.1.529) or a subvariant thereof.
  • the isolated antibody or antigen-binding fragment thereof advantageously binds to both wild-type or original SARS-CoV-2 Spike protein, as well as variants of SARS-CoV-2, such as the Delta strain and/or Omicron strain, thus providing advantages for identifying and treating both the original form and the variants.
  • antibodies disclosed herein have increased specificity and or increased neutralizing activity for the original and or variant forms of SARS-CoV-2, e.g., as compared to other anti-Spike protein antibodies.
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to the Spike protein of a SARS-CoV-2 variant at a level substantially similar to or higher than the level of binding to wild type Spike protein. In some embodiments, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to the Spike protein of a SARS-CoV-2 variant at a level within about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the level of binding to wild type Spike protein.
  • a human monoclonal antibody has the same specificity as a human monoclonal antibody of the disclosure by ascertaining whether the former prevents the latter from binding to SARS-CoV-2. If the human monoclonal antibody being tested competes with the human monoclonal antibody of the disclosure, as shown by a decrease in binding by the human monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies bind to the same, or to a closely relate, epitope.
  • Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the disclosure is to pre-incubate the human monoclonal antibody of the disclosure with the SARS- CoV-2 with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind SARS-CoV-2. If the human monoclonal antibody being tested is inhibited, then in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the disclosure.
  • the disclosure provides antibodies, and antigen-binding fragments thereof, that bind to SARS-CoV-2 Spike protein.
  • the antibodies, or antigen-binding fragment thereof are capable of neutralizing SARS-CoV-2 virus.
  • Neutralizing antibodies bind to virus and prevent infection of cells.
  • an antibody, or antigen-binding fragment thereof, of the disclosure is capable of neutralizing at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 100% of SARS-CoV-2 virus, e.g., in an assay disclosed herein, or in a subject administered with the antibody or antigen-binding fragment thereof.
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, or a fragment thereof, comprising a sequence comprising: a heavy chain with three CDRs comprising the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively and/or a light chain with three CDRs comprising the amino acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively, or a variant thereof comprising less than 8 total amino acid modifications in the CDRs of the antibody, or antigen-binding fragment thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, or a fragment thereof, comprising a sequence comprising: a heavy chain with three CDRs comprising the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 respectively and/or a light chain with three CDRs comprising the amino acid sequences SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 respectively, or a variant thereof comprising less than 8 total amino acid modifications in the CDRs of the antibody, or antigen-binding fragment thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, or a fragment thereof, comprising a sequence comprising: a heavy chain with three CDRs comprising the amino acid sequences SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 respectively and/or a light chain with three CDRs comprising the amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 respectively, or a variant thereof comprising less than 8 total amino acid modifications in the CDRs of the antibody, or antigen-binding fragment thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides an isolated antibody, or an antigen- binding fragment thereof, that binds to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, or a fragment thereof, comprising a sequence comprising: a heavy chain with three CDRs comprising the amino acid sequences SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 respectively and/or a light chain with three CDRs comprising the amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 respectively, or a variant thereof comprising less than 8 total amino acid modifications in the CDRs of the antibody, or antigen-binding fragment thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the antibody, or fragment thereof is a variant comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid modifications in the CDR sequences of the antibody or antigen-binding fragment thereof.
  • the variant comprises less than 7, less than 6, less than 5, less than 4, less than 3, or less than two amino acid modifications in the CDR sequences.
  • the amino acid modifications are amino acid substitutions.
  • the variant comprises less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than two amino acid substitutions in the total CDR sequences present in the antibody or antigen-binding fragment thereof.
  • the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 25, 27, 29 or 31, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E heavy chain.
  • the heavy chain variable region comprises the amino acid sequence set forth in any of SEQ ID NOs: 25, 27, 29 or 31.
  • the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 26, 28, 30 or 32, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E light chain.
  • the light chain variable region comprises the amino acid sequence set forth in any of SEQ ID NOs: 26, 28, 30 or 32.
  • the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 25, 27, 29 or 31, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E heavy chain
  • the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 26, 28, 30 or 32, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E light chain
  • the heavy chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25 and the light chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26.
  • the heavy chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 28.
  • the heavy chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 29 and the light chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 30.
  • the heavy chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 32.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 25 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 26.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32.
  • the antibody, or antigen-binding fragment thereof is humanized.
  • the antibody, or antigen-binding fragment thereof is an IgG antibody, a single chain antibody, a single chain variable fragment (scFv), a univalent antibody lacking a hinge region, a VHH or single-domain antibody (sdAb), or a minibody.
  • the antibody is an IgG antibody comprising a heavy chain comprising an Fc domain, and a light chain.
  • the antibody, or antigen-binding fragment thereof is a VHH or sdAb.
  • the antibody, or antigen-binding fragment thereof is a Fab or a Fab’ fragment.
  • the antibody, or antigen-binding fragment thereof binds to a Spike protein, or a fragment or variant thereof.
  • the disclosure provides isolated antibodies, or antigen-binding fragments thereof, that compete with any one of the antibodies of the disclosure for binding to a Spike protein, or fragment thereof.
  • the Spike protein comprises SEQ ID NO: 43.
  • the Spike protein comprises SEQ ID NO: 44.
  • the Spike protein comprises SEQ ID NO: 45.
  • the isolated antibody, or an antigen-binding fragment thereof binds to the Spike protein with a K d of 50 mM or lower, e.g., a K d having a value less than 50 mM, less than 25 mM, less than 10 mM, less than 5 mM, less than 1 mM, less than 100 nM, or less than 10 nM.
  • the isolated antibody, or an antigen-binding fragment thereof inhibits Spike protein interaction with a human ACE2 receptor.
  • binding of the Spike protein to the human ACE2 receptor is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • binding is inhibited by 75% or greater. Inhibition may be determined, e.g., as described in the accompanying Examples.
  • inhibition of binding of Spike protein to the human ACE2 receptor is determined using a competition ELISA assay, as described in Example 3.
  • the isolated antibody, or an antigen-binding fragment thereof binds an epitope in the receptor binding domain (RBD) of the Spike protein.
  • the epitope is linear or non-linear.
  • the epitope is the same as or overlapping with the region of the Spike protein that binds the human ACE2 receptor.
  • the isolated antibody, or an antigen-binding fragment thereof neutralizes SARS-CoV-2.
  • the isolated antibody, or an antigen-binding fragment thereof inhibits viral and cell membrane fusion.
  • viral and cell membrane fusion is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. Inhibition may be determined, e.g., as described in the accompanying Examples.
  • the ability of the antibody, or antigen-binding fragment thereof, to neutralize SARS-CoV-2 is determined using a lenti-based pseudovirus neutralization assay, as described in Example 4.
  • an antibody, or antigen-binding fragment thereof, of the disclosure neutralizes virus, e.g., SARS-CoV-2 with an IC50 of less than 1000 pM, less than 100 pM, less than 10 pM, or less than 1 pM, e.g., as determined using an assay disclosed herein.
  • virus e.g., SARS-CoV-2 with an IC50 of less than 1000 pM, less than 100 pM, less than 10 pM, or less than 1 pM, e.g., as determined using an assay disclosed herein.
  • heteroconjugate antibodies comprising two covalently joined antibodies.
  • heteroconjugate antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercapto-butyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
  • antibodies of the invention may be modified to enhance the effectiveness of the antibody in neutralizing or preventing viral infection.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.
  • the antibody of the disclosure has modifications of the Fc region, such that the Fc region does not bind to one or more Fc receptors.
  • the Fc receptor is a Fey receptor.
  • antibodies of the disclosure comprise a modified Fc region such that the Fc region does not bind to Fc ⁇ , but still binds to the neonatal Fc receptor.
  • the disclosure provides immunoconjugates comprising an antibody, or antigen binding fragment thereof, of the disclosure conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereol), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereol), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria oficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Illustrative examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutarelde- hyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2, 4-dinitrobenzene).
  • SPDP N-succinimidyl
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987); carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to an antibody (See WO94/11026).
  • MX-DTPA 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
  • coupling a moiety to an antibody may be accomplished by any chemical reaction that can bind the two molecules while enabling the antibody and the moiety to retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • covalent binding is used. Covalent binding can be achieved by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling the antibodies of the disclosure to other molecules.
  • common coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines.
  • organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines.
  • linkers may include: EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene); SPDP (succinimidyl 6-[3(2-pyridyldithio)propionamido]hexanoate); Sulfo-LC-SPDP (Sulfosuccinimidyl 6-(3'-[2- pyridyldithio]-propionamido)hexanoate); and sulfo-NHS (N-hydroxy- sulfo-succinimide) conjugated to EDC.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • SMPT succinimidyloxycarbonyl-alpha-methyl-alpha
  • the linkers described above may generate antibody conjugates with differing physio- chemical properties.
  • sulfo-NHS esters of alkyl carboxylates may be more stable than sulfo-NHS esters of aromatic carboxylates, and NHS-ester containing linkers may be less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.
  • disulfide linkages may be less stable than other linkages because the disulfide linkage may be cleaved in vitro.
  • sulfo-NHS can enhance the stability of carbodiimide couplings.
  • carbodiimide couplings when used in conjunction with sulfo-NHS, may form esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.
  • antibodies, and antigen-binding fragments thereof, of the disclosure can be formulated as immunoliposomes.
  • Immunoliposomes containing the antibody of the disclosure may be prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Patents 4,485,045 and 4,544,545.
  • immunoliposomes with enhanced circulation time may be prepared as disclosed in U.S. Pat. No. 5,013,556.
  • immunoliposomes can be generated by the reverse- phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • Fab’ fragments of the antibody of the disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • the disclosure further includes isolated polypeptides comprising a heavy chain variable region, or variant thereof, disclosed herein, and/or a light chain variable region, or variant thereof, disclosed herein.
  • a variant comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a sequence disclosed herein.
  • the variant comprises all three of the CDRs present in a heavy chain variable region disclosed herein, and/or all three of the CDRs present in a light chain variable region disclosed herein.
  • the variant comprises one or more amino acid modifications, e.g., substitutions, in one or more of the CDRs.
  • a variant comprises three or less, two or less, or one or less modifications in the CDRs of the variant heavy chain variable region and/or three or less, two or less, or one or less modifications in the CDRs of the variant light chain variable region.
  • the disclosure further provides polynucleotide sequences encoding any of the polypeptides disclosed herein, e.g., any antibody heavy chain, or fragment or variant thereof, or any antibody light chain, or fragment or variant thereof.
  • the polynucleotide sequences comprise DNA and/or RNA.
  • the polynucleotide sequences comprise one or more modified RNA sequence.
  • the disclosure further provides vectors comprising a polynucleotide sequence encoding any of the polypeptides disclosed herein, e.g., any antibody heavy chain, or fragment or variant thereof, or any antibody light chain, or fragment or variant thereof.
  • the polynucleotide sequences comprise DNA and/or RNA.
  • the polynucleotide sequences comprise one or more modified RNA sequence.
  • the vectors are plasmids.
  • the vectors are expression vectors, which comprise a promoter operatively linked to the polynucleotide sequence encoding a polypeptide disclosed herein, so as to promote expression of the encoded mRNA and/or polypeptide is a cell.
  • the expression vector is a viral vector.
  • the expression vector is capable of being transferred into a cell, e.g., a mammalian cell, where it expresses the encoded polypeptide.
  • expression vectors may include viral vectors, fusion proteins and chemical conjugates.
  • Viral vectors may include DNA viral vectors and retroviral vectors such as moloney murine leukemia viruses.
  • Illustrative vectors may include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSY) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.
  • HY herpes simplex I virus
  • pox viral vectors can introduce the gene into the cell’s cytoplasm.
  • Avipox virus vectors may result in only a short-term expression of the nucleic acid.
  • Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSY) vectors are used for introducing the nucleic acid into cells.
  • the disclosure further provides a host cell comprising a vector disclosed herein.
  • the host cell may be any of a variety of different cells, including but not limited to, bacterial cells, e.g., E. coli, yeast cells, plant cells, or mammalian cells, e.g., 293 cells. In certain embodiments, such cells are used to propagate the polynucleotide sequence and/or produce the encoded polypeptide.
  • Polynucleotides may be introduced into cells according to a variety of techniques known in the art, such as, e.g., using vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The particular vector chosen may depend upon the target cell and the condition being treated.
  • the introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Additional examples of modes of gene transfer include, but are not limited to, naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.
  • antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which binds primarily the IgG fraction of immune serum.
  • the specific antigen which is the target of the immunoglobulin, or an epitope thereof may be immobilized on a column to purify the immune- specific antibody by immunoaffmity chromatography.
  • Various procedures known in the art may be used for purification of immunoglobulins, for example, the method disclosed by D. Wilkinson (The Computer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
  • the term “monoclonal antibody” or “mAh” or “monoclonal antibody composition”, as used in the disclosure, may refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a light chain and a heavy chain.
  • the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
  • MAbs may contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a mouse, hamster, or other appropriate host animal may be immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent may include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes may then be fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines can be transformed mammalian cells, for example myeloma cells of rodent, bovine and human origin. In some embodiments, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas can include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).
  • the culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose may include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. In some embodiments, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • monoclonal antibodies including the antibodies, and antigen- binding fragments thereof, disclosed herein can be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the DNA can be placed into expression vectors, which are then transfected into host cells, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the disclosure or can be substituted for the variable domains of one antigen-binding site of an antibody of the disclosure to create a chimeric bivalent antibody.
  • the antibodies or antigen-binding fragments thereof as disclosed herein are humanized.
  • the antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains.
  • Epitope binding sites may be wild type or modified by one or more amino acid substitutions.
  • Illustrative methods for humanization of the anti-Fzd antibodies disclosed herein include the methods described in U.S. Patent No. 7,462,697.
  • the humanized antibodies, or antigen-binding fragments thereof comprise not only human-derived constant regions, but the variable regions are also modified, e.g., so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity -determining regions (CDRs) that vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity -determining regions
  • FRs framework regions
  • variable regions can be "reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
  • Application of this approach to various antibodies has been reported by Sato, K., et al., (1993) Cancer Res 53:851-856. Riechmann, L., et al., (1988) Nature 332:323-327 ; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, C.
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody that contains all six CDRs from the mouse antibodies).
  • humanized antibodies have one or more CDRs (e.g., one, two, three, four, five, six, seven or eight) that are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.
  • the antibodies, or antigen-binding fragments thereof, of the present disclosure may be chimeric antibodies or antigen-binding fragments thereof.
  • a chimeric antibody is comprised of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody.
  • the heterologous Fc domain is of human origin.
  • the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.
  • the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes.
  • the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable domain (VL, VH or both).
  • human antibodies may be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome.
  • the human genes may be incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.
  • An animal which provides all the desired modifications may be obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • Xenomouse TM is termed the Xenomouse TM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.
  • scFv single chain Fv
  • J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell may be deleted to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus.
  • the deletion may be made by a targeting vector containing a gene encoding a selectable marker.
  • a transgenic mouse may be produced from the embryonic stem cell, wherein its somatic and germ cells contain the gene encoding the selectable marker.
  • One method for producing an antibody of interest is disclosed in U.S. Pat. No. 5,916,771.
  • This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • antibodies of the disclosure can be expressed by a vector containing a DNA segment encoding a single chain antibody described above.
  • vectors may include, but are not limited to, chemical conjugates such as those described in WO 93/64701, a viral vector (e.g. a DNA or RNA viral vector), plasmids, and phage.
  • a viral vector e.g. a DNA or RNA viral vector
  • plasmids e.g. a DNA or RNA viral vector
  • phage e.g. a viral vector
  • the vectors can be chromosomal, non-chromosomal or synthetic.
  • vectors can be used to express large quantities of antibodies that can be used in a variety of ways.
  • antibodies of the disclosure may be used to detect the presence of SARS-CoV-2 in a sample.
  • Antibodies of the disclosure can also be used to bind to SARS-CoV-2.
  • the disclosure provides methods (also referred to herein as “screening assays”) for identifying antibodies, or fragments or variants thereof, that modulate or otherwise interfere with the fusion of a SARS-CoV-2 virus to the cell membrane.
  • the disclosure also provides methods of identifying antibodies, or fragments or variants thereof, useful to treat SARS-CoV- 2 infection.
  • the disclosure also encompasses antibodies, or fragments or variants thereof, identified using the screening assays described herein.
  • the disclosure provides assays for screening candidate antibodies which modulate the interaction between the SARS-CoV-2 and the cell membrane.
  • the disclosure provides a Competition ELISA method for screening candidate antibodies.
  • the antigen e.g. the spike protein
  • a receptor-Fc fusion protein e.g. a ACE2- Fc fusion protein
  • a wash step may be performed to wash away unbound candidate antibodies and unbound receptor-Fc fusion proteins.
  • An anti-Fc labeled conjugate may then be used as a secondary antibody that can specifically bind to the receptor-Fc fusion protein.
  • the label comprises HRP.
  • the receptor-Fc fusion protein will not be bound to the antigen and may be washed away during the wash step.
  • the anti-Fc secondary antibody can report colorimetrically on the amount of receptor-Fc fusion protein bound complex, thus detecting the presence of candidate antibodies bound to antigens.
  • An illustrative depiction of the method of Competition ELISA is illustrated in FIG. 2.
  • a candidate antibody is introduced to an antibody-antigen complex to determine whether the candidate antibody disrupts the antibody-antigen complex, wherein a disruption of this complex indicates that the candidate antibody modulates the interaction between a SARS-CoV-2 and the cell membrane.
  • At least one SARS-CoV-2 spike protein is provided, which is exposed to at least one neutralizing monoclonal antibody. Formation of an antibody-antigen complex is detected, and one or more candidate antibodies are introduced to the complex. If the antibody-antigen complex is disrupted following introduction of the one or more candidate antibodies, the candidate antibodies may be considered useful to treat a SARS-CoV-2-related disease or disorder.
  • Determining the ability of the candidate antibody to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by coupling the candidate antibody with a radioisotope or enzymatic label such that binding of the candidate antibody to the antigen or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.
  • the candidate antibody can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radio emission or by scintillation counting.
  • the candidate antibody can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting an antibody-antigen complex with a candidate antibody and determining the ability of the test compound to interact with the antigen or otherwise disrupt the existing antibody-antigen complex.
  • determining the ability of the candidate antibody to interact with the antigen and/or disrupt the antibody-antigen complex comprises determining the ability of the candidate antibody to preferentially bind to the antigen or a biologically-active portion thereof, as compared to the antibody.
  • the assay comprises contacting an antibody-antigen complex with a candidate antibody and determining the ability of the candidate antibody to modulate the antibody-antigen complex. Determining the ability of the candidate antibody to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody, in the presence of the candidate antibody.
  • the antibody may be a SARS-CoV-2 neutralizing antibody, such as a monoclonal antibody 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E or any variant thereof.
  • the antigen may be a SARS-CoV-2 spike protein, or a portion thereof.
  • the screening methods disclosed herein may be performed as a cell-based assay or as a cell-free assay.
  • the cell-free assays of the disclosure are amenable to use of both the soluble form or the membrane-bound form of the proteins and fragments thereof. In the case of cell-free assays comprising the membrane-bound forms of the proteins, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the proteins is maintained in solution.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-114, Thesit ® , Isotridecypoly(ethylene glycol ether), N-dodecyl-N,N-dimethyl-3-ammonio-1- propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1 -propane sulfonate (CHAPSO).
  • non-ionic detergents such as n-octylglucoside, n-
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST-antibody fusion proteins or GST-antigen fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the candidate antibody may be incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components.
  • the complexes can be dissociated from the matrix, and the level of antibody- antigen complex formation can be determined using standard techniques.
  • matrices can also be used in the screening assays of the invention.
  • either the antibody or the antigen can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well- known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates.
  • other antibodies reactive with the antibody or antigen of interest can be derivatized to the wells of the plate, and unbound antibody or antigen is trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes in addition to those described above for the GST-immobilized complexes, may include immunodetection of complexes using such other antibodies reactive with the antibody or antigen.
  • a cell-based assay is used as a screening assay and/or to determine the efficacy of a candidate antibody in blocking virus entry into the cell (i.e. neutralization of viral entry).
  • a pseudovirus neutralization assay is used.
  • a pseudovirus displaying the SARS-CoV-2 spike protein its surface is generated using methods known in the art. The pseudovirus retains the ability to bind ACE2, cause membrane fusion and entry into an appropriate human cell but cannot propagate new infectious spike-bearing virions.
  • the pseudovirus comprises HIV-1 genes that constitute backbone and structural elements.
  • the pseudovirus comprises a gene expressing a detectable tag.
  • the detectable tag is expressed using a lentiviral backbone plasmid.
  • a gene expressing a SARS-CoV-2 spike protein may be co-transfected with plasmids encoding the HIV-1 structural genes into 293T cells to generate pseudoviruses.
  • host cells expressing ACE2 on the surface may be used for viral challenge with the pseudovirus.
  • the host cell may be a 293T cell.
  • the candidate antibodies may be co-incubated with the pseudovirus under appropriate conditions, and then overlaid on host cells expressing ACE2.
  • the detectable tag may comprise a fluorescent protein.
  • the detectable tag comprises ZsGreen or Luciferase-IRES-ZsGreen, and detectable Relative light unit (RLU) values can be read on a luminometer. The percent neutralization may be calculated as (RLUvirus - RLUvirus+Ab)/(RLUvirus) X 100.
  • IC50 values may be interpolated from a non-linear regression model [log(agonist) vs response (three parameter)] and represent the Ab concentration at which 50% viral neutralization was recorded. Based on the result of the neutralization assay, candidate antibodies with blocking ability against viral entry may be selected.
  • antibodies selected using any of the screening assays of the disclosure may be further characterized by determining their sequences.
  • Antibody sequencing analysis may be performed using any method known in the art.
  • hybridoma cells expressing the selected antibodies may be used to determine antibody sequence.
  • RNA expressing the selected antibody in hybridoma cells may be extracted using commercially available kits, such as the TRIzol Plus RNA Purification System (Thermo Fisher ® ). The extracted RNA may then be reverse transcribed into cDNA, and the cDNA fragments for the VH and VL domains may be amplified using primers.
  • the amplified sequences may then be cloned into commercially available cloning and selection systems, such as the pCRTM 2.1 vector/One Shot TOPIO system, to generate colonies. The resulting colonies may be screened for the correct size of the gene insert and sequenced. Sequencing methods known in the art may be used. In some embodiments, the sequencing reads may be aligned to obtain a consensus sequence of the clones. This consensus sequence may be analyzed to determine the sequence of the antibody.
  • Methods for screening antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.
  • ELISA enzyme linked immunosorbent assay
  • antibodies, and antigen-binding fragment thereof, disclosed herein may be used for a variety of purposes.
  • antibodies directed against a SARS-CoV-2 spike protein may be used in methods known within the art relating to determining the localization and/or quantification of SARS-CoV-2, for example, for use in measuring levels of the SARS-CoV-2 spike protein within biological samples, for use in diagnostic methods, and for use in imaging the protein.
  • antibodies specific to a SARS-CoV-2 spike protein, or derivative, fragment, analog, homolog, or variant thereof, that contain the antibody derived antigen binding domain are utilized as pharmacologically active compounds (referred to hereinafter as "therapeutics").
  • antibodies specific for a SARS-CoV-2 spike protein can be used to isolate a SARS-CoV-2 polypeptide by standard techniques, such as immunoaffmity, chromatography or immunoprecipitation.
  • An antibody of the disclosure can be used as an agent for detecting the presence of and/or an amount of a SARS-CoV-2 (or a protein or a protein fragment thereol) in a sample, e.g., a biological sample.
  • a sample e.g., a biological sample.
  • antibodies, or antigen-binding fragments thereof are used to determine whether a subject is infected with SARS-CoV-2, e.g., for diagnostic use.
  • Antibodies directed against a SARS-CoV-2 spike protein (or a fragment thereol) can also be used to monitor SARS-CoV-2 or protein levels in a subject’s blood or tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • detecting the presence or absence of a SARS-CoV-2 in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a labeled monoclonal antibody of the disclosure such that the presence of the SARS-CoV-2 is detected in the biological sample.
  • methods include contacting a biological sample obtained from a subject with an antibody, or antigen-binding fragment thereof, disclosed herein, and determining whether the antibody, or antigen-binding fragment thereof bound to any SARS- CoV-2 or Spike protein present in the sample.
  • the antibody, or antigen-binding fragment thereof may be incubated with the biological sample under conditions and for a time sufficient to allow binding of the antibody or fragment thereof to bind to any SARS-CoV-2 present in the biological sample.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the biological sample is blood, a fraction or component of blood including blood serum, blood plasma, or lymph, saliva, nasal fluid, etc.
  • the biological sample is a blood sample, a serum sample, a saliva sample, a mucous sample, a tissue sample, a skin sample, a nasal fluid sample or nasal swap sample, or a urine sample.
  • the biological sample contains virus or protein molecules from the test subject.
  • the biological sample may be a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • Binding may be determined by detecting the presence of complexes comprising the antibody or fragment thereof and the SARS-CoV-2. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, b- galactosidase, and acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/ biotin and avidin /biotin.
  • Suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.
  • a luminescent material includes luminol.
  • Illustrative examples of bioluminescent materials include luciferase, luciferin, and aequorin.
  • Illustrative examples of suitable radioactive material include 125 I, 131 I, 35 S, and 3 H.
  • the antibody contains a detectable label.
  • Antibodies may be polyclonal or monoclonal. In some embodiments, an intact antibody is used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • kits for detecting the presence of a SARS-CoV-2 in a biological sample can comprise: a labeled antibody capable of detecting a SARS-CoV-2 (e.g., an anti-SARS-CoV-2 monoclonal antibody) in a biological sample; means for determining the amount of a SARS-CoV-2 in the sample; and means for comparing the amount of a SARS-CoV-2 in the sample with a standard.
  • the kit can further comprise instructions for using the kit to detect a SARS-CoV-2 in a sample.
  • antibodies and antigen-binding fragments thereof of the disclosure may be used as therapeutic or prophylactic agents. Such agents may be employed to treat, reduce, inhibit or prevent a SARS-CoV-2 infection or related disease or pathology in a subject.
  • an antibody or antigen-binding fragment thereof disclosed herein which have high specificity and high affinity for the SARS-CoV-2 Spike protein, may be administered to the subject and may have an effect due to its binding with the target S protein. Administration of the antibody may abrogate or inhibit or interfere with the internalization of the virus into a cell.
  • the antibody binds to the target and inhibits or prevents SARS- CoV-2 from binding the ACE2 receptor on cells in the subject.
  • the disclosure provides a method of treating, inhibiting, reducing, or preventing a SARS-CoV-2 infection in a subject.
  • the method may also be employed to treat COVID-19 and/or to reduce one or more pathologies associated with SARS-CoV-2 infection, or COVID-19 disease, in a subject, such as, e.g., lung inflammation, cytokine storm, pneumonia and trouble breathing, organ failure, cardiac problems, acute respiratory distress syndrome, blood clots, acute kidney injury, or additional viral and bacterial infections.
  • the method comprises providing to a subject in need thereof an effective amount of an antibody, or antigen-binding fragment thereof, disclosed herein.
  • the subject has been determined to have been infected with SARS-CoV-2, or is considered at risk of being infected with SARS-CoV-2.
  • the subject is diagnosed with COVID-19, the disease resulting from SARS- CoV-2 infection.
  • the subject is provided with a pharmaceutical composition comprising the antibody, or antigen binding fragment thereof.
  • a therapeutically effective amount of an antibody or antigen- binding fragment thereof of the disclosure refers to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, e.g., SARS-CoV-2.
  • the amount required to be administered may depend on the binding affinity of the antibody for its specific antigen and may also depend on the rate at which an administered antibody is depleted from the subject to which it is administered.
  • Illustrative ranges for therapeutically effective dosing of an antibody or antibody fragment of the disclosure may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.
  • illustrative ranges for therapeutically effective dosing of an antibody or antibody fragment of the disclosure may be, by way of nonlimiting example, from about 500 mg to about 2500 mg per single dose.
  • a single dose may comprise about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1500 mg, about 1800 mg, about 2100 mg, or about 2400 mg of an antibody or antibody fragment of the disclosure.
  • Illustrative dosing frequencies may range, for example, from twice daily to once a week.
  • the invention provides methods for preventing or inhibiting a SARS-CoV-2 infection or a related disease or disorder in a subject by administering to the subject an antibody, or fragment or variant thereof, of the disclosure according to the methods of the disclosure.
  • the prophylactic composition may be administered in a pharmaceutical composition.
  • the prophylactic composition may be administered in therapeutically effective amounts.
  • two or more anti- SARS-CoV-2 antibodies are co-administered.
  • administering or “introducing” or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.
  • subjects at high risk of contracting SARS-CoV-2 can be provided with passive immunity to a SARS-CoV-2, according to any of the disclosed methods.
  • Passive immunization may be an effective and safe strategy for the prevention and treatment of viral diseases.
  • Passive immunization using neutralizing human monoclonal antibodies could provide an immediate treatment strategy for emergency prophylaxis and treatment of SARS- CoV-2 infection and related diseases and disorders while the alternative and more time- consuming development of vaccines and new drugs in underway.
  • Subjects at risk for SARS- CoV-2-related diseases or disorders may include patients who have been exposed to or suspected of being exposed to SARS-CoV-2.
  • administration of a prophylactic composition may occur prior to the manifestation of symptoms characteristic of the SARS-CoV -2 -related disease or disorder, such that a disease or disorder is prevented, inhibited, or, alternatively, delayed in its progression.
  • the prophylactic methods of the disclosure may lead to a reduction in the severity or the alleviation of one or more symptoms of a viral infection.
  • the therapeutic or prophylactic composition to be administered is a monoclonal antibody or antigen-binding fragment thereof disclosed herein that neutralizes a SARS-CoV-2.
  • the prophylactic composition of the disclosure can be administered with other antibodies or antibody fragments known to neutralize SARS-CoV-2. Administration of said antibodies can be sequential, concurrent, or alternating.
  • compositions of the disclosure can be administered in conjunction with ancillary immunoregulatory agents, such as cytokines, lymphokines, and chemokines, including, but not limited to, IL-2, modified IL-2 (Cys125 ⁇ Ser125), GM-CSF, IL-12, g-interferon, IP-10, MIRIb, and RANTES.
  • ancillary immunoregulatory agents such as cytokines, lymphokines, and chemokines, including, but not limited to, IL-2, modified IL-2 (Cys125 ⁇ Ser125), GM-CSF, IL-12, g-interferon, IP-10, MIRIb, and RANTES.
  • the therapeutic or prophylactic composition is administered prior to diagnosis of the infection. In some embodiments, the therapeutic or prophylactic composition is administered after diagnosis. Efficaciousness of the therapeutic or prophylactic composition may be determined in association with any known method for diagnosing or treating the particular disorder or infection.
  • the disclosure provides methods of treating a SARS-CoV-2 infection or related disease or disorder in a patient.
  • the method involves administering a therapeutic composition that neutralize the SARS-CoV-2 to a patient infected with SARS-CoV-2 or suffering from the related disease or disorder, such as COVID-19.
  • the therapeutic composition may be administered in therapeutically effective amounts.
  • two or more anti-SARS-CoV-2 antibodies are co- administered.
  • treatment means obtaining a desired pharmacologic and/or physiologic effect.
  • Preventing” or “inhibiting” a disease or disorder may be prophylactic in terms of completely or partially preventing or inhibiting a disease, infection, or symptom thereof, e.g., reducing the likelihood that the disease, infection, or symptom thereof occurs in the subject, and “treatment” may be therapeutic in terms of a partial or complete cure for a disease, infection, and/or adverse effect attributable to the disease or infection.
  • Prevention and “treatment” as used herein are applicable to any treatment of a disease or disorder (e.g., an infection, such as a SARS-CoV2 infection) in a mammal, and includes: (a) preventing the disease or disorder from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease or disorder, i.e., arresting its development or inhibiting its progression; or (c) relieving the disease or disorder, i.e., causing regression of the disease or disorder or improvement of one or more symptoms of the disease.
  • a disease or disorder e.g., an infection, such as a SARS-CoV2 infection
  • a mammal includes: (a) preventing the disease or disorder from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease or disorder, i.e., arresting its development or inhibiting its progression; or (c) relieving the disease or disorder,
  • treat refers to stasis or a postponement of development of one or more symptoms associated with a disease or disorder described herein, or ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, or ameliorating or preventing the underlying metabolic causes of symptoms.
  • beneficial result has been conferred on a mammalian subject with a disease or symptom, or with the potential to develop such disease or symptom.
  • a response is achieved when the patient experiences partial or total alleviation, or reduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival.
  • the expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors.
  • the therapeutic agent may be administered before, during or after the onset of disease or disorder.
  • the treatment of ongoing disease or disorder, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
  • the subject therapy may be administered during the symptomatic stage of the disease or disorder, and in some cases after the symptomatic stage of the disease.
  • administration of a therapeutic composition may occur after the manifestation of symptoms characteristic of the SARS-CoV-2 infection or related disease or disorder, such that a disease or disorder is delayed in its progression and/or reduced in its severity.
  • administration of a therapeutic composition may occur after diagnosis of SARS-CoV-2 infection or COVID-19, such that the infection or a disease or disorder is delayed in its progression or reduced in its severity.
  • the therapeutic methods of the disclosure may lead to a reduction in the severity or the alleviation of one or more symptoms of COVID-19.
  • the therapeutic composition can be administered with other antibodies or antibody fragments known to neutralize SARS-CoV-2. Administration of said antibodies can be sequential, concurrent, or alternating.
  • therapeutic compositions of the disclosure can be administered in conjunction with ancillary immunoregulatory agents, such as cytokines, lymphokines, and chemokines, including, but not limited to, IL-2, modified IL-2 (Cys125 ⁇ Ser125), GM-CSF, IL-12, g-interferon, IP-10, MIRIb, and RANTES.
  • ancillary immunoregulatory agents such as cytokines, lymphokines, and chemokines, including, but not limited to, IL-2, modified IL-2 (Cys125 ⁇ Ser125), GM-CSF, IL-12, g-interferon, IP-10, MIRIb, and RANTES.
  • the therapeutic composition is administered prior to diagnosis of the infection.
  • the prophylactic composition is administered after diagnosis. Efficaciousness of the prophylactic composition may be determined in association with any known method for diagnosing or treating the particular disorder or infection.
  • the disclosure provides methods for treating, inhibiting, reducing, or preventing a SARS-CoV-2-related disease or disorder in a patient by administering an antibody of the disclosure, such as 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E, or fragments or variants thereof, with an anti-viral agent known in the art.
  • an antibody of the disclosure such as 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E, or fragments or variants thereof.
  • an anti-viral agent known in the art.
  • anti-viral agents include peptides, nucleic acids, small molecules, inhibitors, and RNAi.
  • Antibodies specifically binding a SARS-CoV-2 spike protein or a fragment thereof of the disclosure, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment, inhibition, or prevention of SARS-CoV-2 infection and related disorders in the form of pharmaceutical compositions.
  • Principles and considerations involved in preparing such pharmaceutical compositions, as well as guidance in the choice of components are provided, by way of non-limiting example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub.
  • compositions comprising one or more antibodies, or fragments or variants thereof, of the disclosure.
  • Pharmaceutical compositions may include prophylactic compositions and therapeutic compositions.
  • the prophylactic compositions can be used to prevent a SARS-CoV-2 infection and the therapeutic compositions can be used to treat individuals following a SARS-CoV-2 infection.
  • compositions of the disclosure may comprise antibodies, or fragments or variants thereof, identified by a screening assay disclosed herein and/or an antibody identified according to the methods disclosed.
  • pharmaceutical compositions of the disclosure may comprise monoclonal antibody 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or417.116E, or fragments or variants thereof.
  • the pharmaceutical composition comprises one or more of the antibodies, or antigen-binding fragments thereof, disclosed herein, or any of the related polypeptides disclosed herein.
  • compositions of the disclosure may have superior immunotherapeutic properties over other SARS-CoV-2 vaccines and/or therapeutic compositions.
  • compositions suitable for administration may comprise the antibody or agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Illustrative examples of suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • the pharmaceutical compositions are stable under the conditions of manufacture and storage.
  • the pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • the pharmaceutical composition includes isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation may include vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser.
  • Said container or dispenser may contain a suitable propellant, e.g., agas such as carbon dioxide, or anebulizer.
  • systemic administration can be performed by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated may be used in the formulation.
  • penetrants may include detergents, bile salts, and fusidic acid derivatives.
  • transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art.
  • compositions of the disclosure comprise antibodies that include the Fc region.
  • pharmaceutical compositions of the disclosure comprise antibody fragments that lack the Fc region. Such fragments may retain the ability to bind the target protein sequence and can be synthesized chemically and/or produced by recombinant DNA technology.
  • the pharmaceutical composition can also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or a growth-inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or a growth-inhibitory agent.
  • cytokine cytokine
  • chemotherapeutic agent a growth-inhibitory agent.
  • active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization.
  • microcapsules include hydroxymethylcellulose microcapsules, gelatin-microcapsules, poly-(methylmethacrylate) microcapsules, liposomes, albumin microspheres, microemulsions, nano-particles, nanocapsules, and macroemulsions.
  • the formulation to be used for in vivo administration is sterile. This may be accomplished by filtration through sterile filtration membranes.
  • oral or parenteral compositions are formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure may be dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Spike protein or a fragment thereof, comprising a sequence comprising:
  • a heavy chain with three CDRs comprising the amino acid sequences SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 respectively and/or a light chain with three CDRs comprising the amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 respectively; or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid modifications in said CDR sequences.
  • the isolated antibody, or antigen-binding fragment thereof, of embodiment 1, comprising a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any of SEQ ID NOs: 25, 27, 29 or 31, optionally comprising the three CDRs of the 417.19C, 417.53C, 417.56B, 417.64E, 417.66D, 417.91G, or 417.116E heavy chain.
  • a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier, and a therapeutically effective amount of the isolated antibody, or antigen-binding fragment thereof, according to any of embodiments 1-20.
  • a method for preventing, inhibiting, or treating a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection comprising administering to a subject a composition comprising a therapeutically effective amount of the isolated antibody, or antigen- binding fragment thereof, according to any one of embodiments 1-20.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • [0200] 28 The method of any one of embodiments 25-27, wherein the method further comprises administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor.
  • composition comprises two or more antibodies specific to SARS-CoV-2.
  • a method of preventing, inhibiting, or treating a disease or disorder associated with a SARS-CoV-2 infection comprising administering to a subject a composition comprising a therapeutically effective amount of the isolated antibody, or antigen-binding fragment thereof, of any one of embodiments 1-20.
  • composition comprises two or more antibodies specific to SARS-CoV-2.
  • a method of determining if a subject is infected with SARS-CoV-2 comprising: contacting a biological sample obtained from the subject with the isolated antibody, or antigen-binding fragment thereof, of any one of embodiments 1-20; determining an amount of the antibody, or antigen-binding fragment thereof, bound to the biological sample, thereby determining the presence or absence of SARS-CoV-2 in the biological sample, thus determining if the subject is infected with SARS-CoV-2.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) greater than an amount or level described herein.
  • An “increase” may be an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 2-fold, at least 5-fold, or at least 10-fold.
  • a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) less than an amount or level described herein, for example an amount that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of an amount or level described herein.
  • a decrease may be a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, least 80%, at least 90%, or about 100%.
  • an increase or decrease is relative to a value determined prior to infection or prior to treatment.
  • an increase or decrease is relative to a predetermined value, e.g., an average value obtained from numerous subjects, e.g., prior to infection or prior to treatment.
  • a “composition” can comprise an active agent, e.g., an immunogenic polypeptide, and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient.
  • a composition may be a pharmaceutical composition.
  • the compositions are sterile, substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • a polynucleotide or polypeptide has a certain percent " sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • sequence identity refers, with respect to a polypeptide or polynucleotide sequence, to the percentage of exact matching residues in an alignment of that “query” sequence to a “subject” sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated, unless specified otherwise, across the full length of the subject sequence.
  • a query sequence “shares at least x% identity to” a subject sequence if, when the query sequence is aligned to the subject sequence, at least x% (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence.
  • the subject sequence has variable positions (e.g residues denoted X)
  • an alignment to any residue in the query sequence is counted as a match.
  • Sequence similarity can be determined in a number of different manners.
  • sequences can be aligned using the methods and computer programs, including the NCBI Blast service (e.g., BLAST+ version 2.12.0), available over the worldwide web at ncbi.nlm.nih.gov/BLAST/, which is used unless indicated to the contrary.
  • NCBI Blast service e.g., BLAST+ version 2.12.0
  • FASTA available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed.
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.
  • mutant refers to a nucleotide sequence, e.g., gene, or gene product, e.g., RNA or protein, that is present in a wild-type cell, tissue, organ or organism, e.g., virus.
  • variant or “mutant” as used herein encompasses a mutant of a reference polynucleotide or polypeptide sequence, for example a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence.
  • a variant or mutant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence.
  • a variant or mutant may be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length native polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence.
  • a variant or mutant may be a polypeptide having a sequence identity of 70% or more with a full-length native polypeptide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full- length native polypeptide sequence.
  • Variants may also include variant fragments of a reference, e.g., native, sequence sharing a sequence identity of 70% or more with a fragment of the reference, e.g., native, sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence.
  • variant cells, tissues, organs, or organisms comprising the variant polynucleotide or polypeptide.
  • a “subject,” “individual,” or “patient” as used in the disclosure includes any animal that exhibits a symptom of a condition that can be detected or identified with compositions of the disclosure. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals (such as horses, cows, sheep, pigs), and domestic animals or pets (such as a cat or dog). In some embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate and, in some embodiments, the subject is a human.
  • any method described herein may be rewritten into Swiss-type format for the use of any agent described herein, for the manufacture of a medicament, in treating any of the disorders described herein.
  • any method described herein to be rewritten as a compound for use claim, or as a use of a compound claim.
  • EXAMPLE 1 GENERATING S-SPECIFIC HYBRIDOMAS [0233] Neutralizing monoclonal antibodies (mAbs) against SARS-CoV-2 Spike protein (S) were raised by immunizing C57BL/6 mice with S protein. Mice expressing high titers of anti- S antibodies were selected for hybridoma generation to express Abs for subsequent screening. Hybridomas were generated by fusing sp2/mIL-6 myeloma cells with splenic cells harvested from selected mice in the presence of PEG.
  • mAbs SARS-CoV-2 Spike protein
  • EXAMPLE 2 CHARACTERIZATION AND PURIFICATION OF S-SPECIFIC ANTIBODIES
  • the isotypes of the 21 selected hybridoma clones from Example 1 were determined. Supernatants from two rounds of cloning were collected for characterizing the Abs. Light chain locus identity and isotype of the Abs of the 21 primary clones are listed in Table 1. In all, three Abs of isotype IgG1 and nine each of isotypes IgG2a and IgG2b were obtained. Except for four Abs, all others contained the Kappa light chain (Table 1). For all 21 antibodies, purity and yield were in the expected range and binding to recombinant S was confirmed using ELISA and IRIS (Interferometric Reflectance Imaging Sensor).
  • Abs from the 21 primary hybridoma clones were purified from 250mL of low-density cell cultures by Protein A affinity chromatography (MabSelect PrismA lmL column, Cytiva). Briefly, supernatants containing the antibodies of interest were applied over the column. mAbs bound to the resin were washed with phosphate buffered saline (PBS) and eluted using an acidic gradient of 0.1M citrate (pH 5.0-3.0). The eluted fractions containing the antibodies of interest were neutralized with 1M Tris and dialyzed against PBS. Purified antibodies were concentrated to 1-3 mg/ml, and the concentration was determined by absorbance at 280nm using a mass extinction coefficient of 1.35 mg-1cm-1.
  • PBS phosphate buffered saline
  • EXAMPLE 3 ANTIBODY INHIBITION OF S-ACE2 INTERACTION
  • the method of Competition ELISA is illustrated in FIG. 2 and described as follows: a mammalian-codon-optimized gene encoding human ACE2 residues 1-615 C-terminally fused to a human IgG Fc region was synthesized and subcloned into the eukaryotic expression vector pcDNA3.1(+)-Hygro.
  • the nucleic acid sequence encoding the ACE2-Fc fusion protein is disclosed in SEQ ID NO: 41
  • the amino acid sequence of the ACE2-Fc fusion protein is disclosed in SEQ ID NO: 42.
  • Expi-CHO cells were transfected with said vector harboring the ACE2 gene, and cultures were harvested 10-14 days later.
  • the secreted protein was purified from the supernatant using Protein A affinity chromatography.
  • S was coated on 96-well plates at 2 ⁇ g/ml overnight followed by blocking (Inblock, InBios). The wells were incubated for 1 hour with a mixture of the ACE2Fc (lOOng/ml) and the purified Ab in a series of concentrations ranging from 8-0.5 ⁇ g/ml. Plates were washed to remove unbound material. An anti-human FC-HRP conjugate was then used as a secondary antibody which can specifically bind to ACE2Fc stably associated with the S coated on the wells. An anti Fc-HRP secondary antibody reported colorimetrically on the amount of S-ACE2Fc bound complex in a well.
  • Binding inhibition was calculated based on the color intensity of S bound to 100 ng/mL of ACE2Fc in the presence or absence of Abs. Percent binding inhibition was thus calculated as ((OD 450nm No Ab - OD 450nmAb/ OD 45 nmNo Ab) * 100).
  • the 21 purified Abs were tested for neutralization of viral entry into ACE2 expressing cells by using them in a luciferase-tagged, SARS-CoV-2 Spike-expressing-pseudo virus- mediated cell entry assay. This assay recapitulates the initial steps in the infectivity of SARS- CoV-2 and is considered the confirmatory assay for viral neutralization. Neutralization of viral cell entry was accomplished by blocking the binding between viral S and cell surface ACE2, and the resulting degree of viral infection was measured as intracellular luciferase signal intensity.
  • a lentiviral-based pseudovirus neutralization assay was used.
  • the full-length SARS-CoV-2 (Wuhan 1 strain) S gene was co-transfected with plasmids encoding the HIV-1 structural genes into 293T cells stably expressing human ACE2, using PEI Max and grown at 32°C for 3 days prior to harvesting supernatants containing pseudoviruses.
  • the resulting pseudovirus displays S on the surface of the virion, whereas the HIV-1 genes constitute the backbone and structural elements. It retains the ability to bind ACE2, cause membrane fusion and entry into an appropriate human cell but cannot propagate new infectious spike-bearing virions.
  • 293T cells expressing ACE2 were plated in 96-well plates at a density of 10 4 / well 24 hours prior to viral challenge, and polybrene was added at 2 ⁇ g/ml for 30 min prior to viral challenge.
  • the Abs were serially diluted (10- 1.3X10 -6 ⁇ g/ml) and co-incubated with the pseudovirus for 60 min at 37°C and then overlaid on 293T/ACE2 cells. The plates were incubated at 37°C for 72 hours, the media removed, and 100 pi of Steady Glo (Promega) was added.
  • Relative light unit (RLU) values were read on a luminometer, and the percent neutralization was calculated as ( RLUvirus - RLUvirus+Ab)/( RLUvirus) X 100.
  • IC50 values were interpolated from a non-linear regression model [log(agonist) vs response (three parameter)] and represent the Ab concentration at which 50% viral neutralization was recorded.
  • IC50 values ranged from 0.139 ⁇ g/ml to 8.8 X 10 -5 ⁇ g/ml (Table 2), or 926.67 pM to 0.587 pM. Except for 417.53C, the six other Abs displayed an IC50 in the low picomolar concentrations (Table 2). Thus, the highly confirmatory pseudovirus neutralization assay confirmed that the Abs that inhibited S-ACE2Fc binding were proficient in neutralizing pseudovirus entry into cells, potentially preventing SARS-CoV-2 infection.
  • EXAMPLE 5 ANTIBODY SEQUENCING ANALYSIS
  • the seven Abs that were selected based on their ability to neutralize S-ACE2 binding by both competition ELISA and pseudovirus neutralization assay were prepared for cloning and sequencing using standard 5’ RACE amplification, followed by cloning and selection for sequence determination.
  • Total RNA was isolated from frozen hybridoma cell lysates using the TRIzol Plus RNA Purification System (Thermo Fisher ® ) per manufacturer’s instructions. Total RNA was then reverse transcribed into cDNA using isotype-specific anti-sense primers based on the isotype of the Ab.
  • VH and VL The fragments for the variable region for both heavy and light chains, VH and VL, were amplified using isotype-specific primers (InBios) and the 5’ RACE System for Rapid Amplification kit (Thermo Fisher ® ). Amplified sequences were then selected after cloning and screening of colonies using the pCRTM 2.1 vector/One Shot TOPIO cloning and selection system. At least five colonies with inserts of correct sizes were sequenced for each fragment. The sequences were then aligned to obtain a consensus sequence of the clones. The VH and VL were analyzed using NCBI IgBLAST for variable domain sequences and SignalP- 5.0 for presence and prediction of signal peptide location of their cleavage sites.
  • SEQ ID NO: 37 1 atggctgtcc tggcgctact cctctgcctg gtgactttcc caagctgtgc cctgtcccag

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Abstract

La divulgation concerne des anticorps qui se lient au SARS-CoV-2 et des méthodes d'utilisation de ceux-ci. Les anticorps divulgués peuvent être utilisés pour traiter ou prévenir des infections par le SARS-CoV-2 et des troubles apparentés, et pour déterminer si un sujet est infecté par le SARS-CoV-2.
PCT/US2022/025560 2021-04-20 2022-04-20 Anticorps neutralisants contre le sars-cov-2 WO2022226079A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090169547A1 (en) * 2005-11-24 2009-07-02 Ugur Sahin Monoclonal antibodies against claudin-18 for treatment of cancer
US20140057302A1 (en) * 2012-08-21 2014-02-27 Janssen Pharmaceutica Nv Antibodies to Risperidone and Use Thereof
WO2021045836A1 (fr) * 2020-04-02 2021-03-11 Regeneron Pharmaceuticals, Inc. Anticorps anti-glycoprotéine spike du sars-cov 2 et fragments de liaison à l'antigène de ceux-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090169547A1 (en) * 2005-11-24 2009-07-02 Ugur Sahin Monoclonal antibodies against claudin-18 for treatment of cancer
US20140057302A1 (en) * 2012-08-21 2014-02-27 Janssen Pharmaceutica Nv Antibodies to Risperidone and Use Thereof
WO2021045836A1 (fr) * 2020-04-02 2021-03-11 Regeneron Pharmaceuticals, Inc. Anticorps anti-glycoprotéine spike du sars-cov 2 et fragments de liaison à l'antigène de ceux-ci

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